The continuous and intensive use of insecticides against insect pests has resulted in the development of resistant strains. Resistance to cyclodiene insecticides accounts for more than 60% of reported cases of insecticide resistance, and has been documented in at least 277 species. ffrench-Constant et al., Proc. Natl. Acad. Sci. USA 90:1957 (1993). For example, several years of selection by dieldrin residual spraying for malaria control resulted in the resistance factor becoming a predominant characteristic of the Anopheles gambiae population, with a frequency of 90%. Metcalf and Flint, Destructive and Useful Insects, Their Habits & Control, 4th ed., (McGraw-Hill, New York, 1962)(p. 397).
Studies of insecticide resistance in more than 30 species indicate that only a limited number of independent mechanisms are involved. Many different classes of insecticides either attack the same target site, and/or are metabolized by the same degradative enzymes. ffrench-Constant et al., in: Molecular Approaches to Fundamental and Applied Entomology, (Springer-Verlag, New York, 1992) (pp. 1-37).
Despite this apparent conservatism in resistance mechanisms, and the continued design of many insecticides around targets in the insect nervous system, resistance is still poorly understood at the molecular level. This is due at least in part to the fact that most of the toxicological and biochemical studies on resistance have been done in pest species where comprehensive genetic and molecular studies are difficult to perform.
Current strategies to combat resistance often rely on the use of alternative compounds. However, the lack of understanding of the basis of resistance results in this strategy being one of trial and error. Compound analogs are simply put to the test in the field, without any significant prescreening for efficacy.
Thus, what is needed is an easy, reliable method to determine the safety and efficacy of newly developed insecticides. The method should be amenable to screening insecticides in an in vitro assay prior to any field testing. Since large numbers of compounds (e.g., compound libraries) need to be evaluated, this method should be such that automation is feasible.
DEFINITIONS
The term "pesticide" as used herein refers to any type of chemical compound which has lethal or sub-lethal effects on eukaryotic cells. Encompassed within "pesticide" are such compounds as insecticides, vermicides, mutagens, carcinogens, and any other compound useful for killing, mutating, or debilitating organisms such as insects. "Pesticides" include, but are not limited to polychlorinated hydrocarbons (such as DDT [dichlorodiphenyltrichloroethane] dieldrin, aldrin, chlordane and lindane) organophosphorous compounds, carbamates, and any other compound useful in the killing of pests.
The term "cyclodiene" refers to any chemical compound comprised of a cyclic member which contains two double bonds. "Cyclodiene" includes, but is not limited to aldrin, endrin, endossulfan, lindane and derivatives or metabolites thereof (e.g., dieldrin).
The term "drug" as used herein, refers to any medicinal substance used in humans or other animals. Encompassed within this definition are compound analogs, naturally occurring, synthetic and recombinant pharmaceuticals, hormones, antimicrobials, neurotransmitters, etc.
As used herein, the term "neurotransmitter" includes any compound which functions in the nervous system to result in the transmission of chemical signals between cells. Encompassed within this definition are substances released from the axon terminal of a presynaptic neuron on excitation, which diffuses across the synaptic cleft to either excite or inhibit the synaptic cleft to either excite or inhibit the target cell. False neurotransmitters are also included (e.g., a compound that can be released from presynaptic vesicles but that has little effect on postsynaptic receptors). Examples of neurotransmitters include, but are not limited to neuropeptides, acetocholine, and amino acids (e.g., GABA). Other compounds are also contemplated, including dopamine, norepinephrine, etc.
The term "permeant" refers to molecules which are capable of entering cells by means of ion channels or other mechanisms. "Permeant" includes, but is not limited to ions such as chloride, potassium, sodium, and thiocyanate.
The term "toxin" refers to any compound or molecule which is capable of causing cell death upon entering cells by means such as ion channels or other mechanisms. "Toxins" include, but are not limited to such compounds as thiocyanates.
The term "GABA" refers to .gamma.-aminobutyric acid, a major inhibitory neurotransmitter in both vertebrates and invertebrates. Kuffler and Edwards, J. Neurophysiol., 21:589 (1965) Otsuka et al., Proc. Natl. Acad. Sci USA 56:1110 (1966); Usherwood and Grundfest, J. Neurophysiol., 28:497 (1965).
The term "GABA receptors" thus refers to structures expressed by cells and which recognize GABA. In vertebrates, several subunits of different types (.alpha., .beta., .gamma., .delta., or .rho.) are assembled in an unknown stoichiometry to form a GABA-gated Cl.sup.- channel termed the GABA receptor. Olsen and Tobin, FASEB J. 4:1469 (1990). In invertebrates, only two GABA receptor subunits have been identified to date--a GABA.sub.A .beta. subunit homologue from the mollusc (Lymnaea stagnalis) (Harvey et al., EMBO J. 10:3239 (1991), and a novel subunit termed Rdl (from a mutant, resistant to dieldrin) from Drosophila melanogaster. In the fly, the Rdl subunit forms functional channels as a homomultimer. ffrench-Constant, Nature, in press.
The term "agonist" refers to molecules or compounds which mimic the action of a "native" or "natural" compound. Agonists may be homologous to these natural compounds in respect to conformation, charge or other characteristics. Thus, agonists may be recognized by receptors expressed on cell surfaces. This recognition may result in physiologic and/or biochemical changes within the cell, such that the cell reacts to the presence of the agonist in the same manner as if the natural compound was present.
The term "antagonist" refers to molecules or compounds which inhibit the action of a "native" or "natural" compound. Antagonists may or may not be homologous to these natural compounds in respect to conformation, charge or other characteristics. Thus, antagonists may be recognized by the same or different receptors that are recognized by an agonist (e.g., GABA). Antagonists may have allosteric effects which prevent the action of an agonist (e.g., prevent opening of the chloride ion channel). Or, antagonists may prevent the function of the agonist (e.g., by blocking the passage of chloride ions in the channels). In contrast to the agonists, antagonistic compounds do not result in physiologic and/or biochemical changes within the cell such that the cell reacts to the presence of the antagonist in the same manner as if the natural compound was present.
Picrotoxinin (PTX) is an example of a GABA antagonist which is recognized by the GABA receptor. "Picrotoxinin" is defined as the toxin component within "picrotoxin" (cocculin), an intensely bitter and very poisonous alkaloid isolated from the seed of Anamirta cocculus. Picrotoxin is a central nervous and respiratory system stimulant which may be used as a veterinary antidote to barbiturates. Budavari et al., (eds.), Merck Index (Merck & Co., Rahway, N.J., 1989) (p. 1177).
The term "host cell" or "cell" refers to any cell which is used in any of the screening assays for detection of resistance. "Host cell" or "cell" also refers to any cell which either naturally expresses particular receptors of interest or is genetically altered so as to produce these normal or mutated receptors.
As used herein, the term "diagnosis" refers to the determination of whether an insect or other organism is susceptible or resistant to a particular pesticide or other compound.
The term "hybridization" as used herein refers to the formation of sequence-specific, base-paired duplexes from any combination of nucleic acid fragments. Hybridization, regardless of the method used, requires some complementarity between the sequence of interest (the target sequence) and the fragment of nucleic acid used to detect the target sequence and/or perform the test (e.g., the probe). Thus, these duplexes may be completely complementary or may include mismatched sequences.
For example, where it is desired to detect simply the presence or absence of insect DNA or RNA, it is only important that the hybridization method ensures hybridization when the relevant sequence is present; conditions can be selected where both partially complementary probes and completely complementary probes will hybridize. However, other diagnostic applications may require that the method of hybridization distinguish between variant target sequences. For example, it may be of interest that a particular allelic variant is present.
Methods have been devised to enable discrimination between partial and complete complementarity. One approach is to take advantage of the temperature requirements of the specific hybridization under study. In typical melting curve experiments, such as those described by Wallace et al., Nucl. Acids Res., 6:35632 (1979) and Nucl. Acids Res. 9:879 (1981), it is observed that partially complementary probe-target duplexes display a lower thermal stability than do completely complementary probe-target duplexes. The best estimate is that a 1% mismatch causes a reduction in the thermal stability of duplexes, as measured by the duplex melting temperature (T.sub.m), by 1.degree. C. See R. J. Britten and E. H. Davidson, in: Nucleic Acid Hybridisation, (B. D. Hames and S. J. Higgins, eds) (IRL Press, Washington, 1985)(pp. 3-15). The T.sub.m is also affected by the length of the base-paired region of a duplex, according to the equation D=500/L, wherein D is the reduction in T.sub.m (0.degree. C.) and L is the length of the base-paired duplex. The base composition of the duplex is another factor which affects its stability. In normal salt solutions, GC base pairs are more stable than AT pairs, thus the T.sub.m of a particular duplex is related to its GC content according to the equation T.sub.m =0.41(% GC)+69.3. As used herein, the terms "substantial complementarity" or "substantially complementary" refer to a nucleic acid duplex in which the T.sub.m is within (plus or minus) 10.degree. C. of the T.sub.m of a completely complementary duplex.
There are various applications that may require that the hybridization method distinguish between partial and complete complementarity. For example, it may be of interest to detect genetic polymorphisms. As used herein, the term "genetic polymorphisms" refers to the condition in which two or more different nucleotide sequences coexist in the same interbreeding population in a DNA sequence. In many instances, it is desirable to combine hybridization with other techniques (such as restriction enzyme analysis).
The term "probe" as used herein refers to a nucleic acid sequence which is used to detect the presence of a complementary sequence by hybridization.
The term "primer" as used herein refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.
As used herein, the term "target" refers to the region of nucleic acid bounded by the primers used for polymerase chain reaction. Thus, the "target" is sought to be sorted out from other nucleic acid sequences. A "segment" is defined as a region of nucleic acid within the target sequence.
As used herein, the term "polymerase chain reaction" ("PCR") refers to the method of K. B. Mullis U.S. Pat. Nos. 4,683,195 and 4,683,202, hereby incorporated by reference, which describe a method for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. This process for amplifying the target sequence consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to their respective strands of the double stranded target sequence. To effect amplification, the mixture is denatured and the primers then annealed to their complementary sequences within the target molecule. Following annealing, the primers are extended with a polymerase so as to form a new pair of complementary strands. The steps of denaturation, primer annealing and polymerase extension can be repeated many times (i.e., denaturation, annealing and extension constitute one "cycle"; there can be numerous "cycles") to obtain a high concentration of an amplified segment of the desired target sequence. The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of the repeating aspect of the process, the method is referred to as the "polymerase chain reaction" (hereinafter "PCR"). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be "PCR amplified".
With PCR, it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of .sup.32 P-labeled deoxynucleotide triphosphates, such as dCTP or DATP, into the amplified segment). In addition to genomic DNA, any oligonucleotide sequence can be amplified with the appropriate set of primer molecules. In particular, the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications.
As used herein, the term "PCR product" refers to the resultant mixture of compounds after two or more cycles of the PCR steps of denaturation, annealing and extension are complete. "PCR product" encompasses the case where there has been amplification of one or more segments of one or more target sequences.
As used herein, the term "nested primers" refers to primers that anneal to the target sequence in an area that is inside the annealing boundaries used to start PCR. K. B. Mullis, et al., Cold Spring Harbor Symposia, Vol. LI, pp. 263-273 (1986). Because the nested primers anneal to the target inside the annealing boundaries of the starting primers, the predominant PCR-amplified product of the starting primers is necessarily a longer sequence, than that defined by the annealing boundaries of the nested primers. The PCR-amplified product of the nested primers is an amplified segment of the target sequence that cannot, therefore, anneal with the starting primers.
As used herein, the term "amplification reagents" refers to those reagents (deoxyribonucleoside triphosphates, buffer, etc.), needed for amplification except for primers, nucleic acid template and the amplification enzyme.
As used herein, the terms "restriction endonucleases" and "restriction enzymes" refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.
As used in the present invention, the term "transformation" refers to the introduction of foreign genetic material into a cell or organism. Transformation may be accomplished by any method known which permits the successful introduction of nucleic acids into cells and which results in the expression of the introduced nucleic acid. For example, transformation may be used to introduce cloned DNA encoding a normal or mutant GABA receptor into a cell which normally does not express this receptor.
"Transformation" includes but is not limited to such methods as transfection, microinjection, electroporation, and lipofection (liposome-mediated gene transfer). Transformation may be accomplished through use of any expression vector. For example, the use of baculovirus to introduce foreign nucleic acid into insect cells is contemplated. The term "transformation" also includes methods such as P-element mediated germline transformation of whole insects.
"Field organisms" are insects or other organisms collected from their natural or an artificial habitat. For example, mosquitoes may be collected from bodies of water, near agricultural areas, or from insect traps. Such organisms may also be collected from the bodies of other animals (e.g., mosquitoes removed from the skin of chickens used as viral encephalitis sentinel animals) or from vegetation. Field organisms include any organisms collected and isolated from an environment other than the laboratory setting.
As used in the present invention, the term "beneficial organisms" refers to insects or other animals which provide benefits to successful agriculture. These insects may act as predators of insects which are known to be harmful to crops. In this case, the predatory beneficial insects use the harmful insects as a food source. The term also encompasses organisms which kill harmful insects but do not utilize the harmful insects as a food source.
As used in the present invention, the term "harmful insects" refers to insect species or varieties which are destructive to agriculture (e.g., farming), are vectors or hosts of disease-causing microorganisms (including parasites), or cause harm to flora or fauna. The term encompasses "pest" insects including, but not limited to such insects as cockroaches, flies, spiders, etc. It also encompasses insects such as mosquitoes, beetles, sandflies, ticks and any other members of the insect family which cause harm to animals or plants.
The term "sample" as used herein refers to any type of material obtained from any organism, including humans, insects or other animals (e.g., any bodily fluid or tissue), cell or tissue cultures, cell lines, primary cell cultures. "Sample" also encompasses any of these cells which naturally express normal or mutant GABA receptors, and cells which have been transformed to express normal or mutant GABA receptors. Such transformed cells may transiently or permanently express normal or mutant GABA receptors.